1. Field of the Invention
The present invention relates to methods for efficient removal of a dibenzofulvene amine adduct generated as a by-product during deprotection of an amino acid compound protected with an Fmoc (9-fluorenylmethoxycarbonyl) group and the like.
2. Discussion of the Background
The Fmoc group is an amino-protecting group and is widely used since it provides many advantages such as suitable liposolubility imparted to the protected compound to facilitate handling, easy tracking of reaction utilizing imparted UV absorption, stability in the neutral to acidic range, easy deprotection under mild reaction conditions with amine and the like. Particularly, it is an important protecting group of amino group of amino acid and peptide in peptide synthesis.
During removal of the Fmoc group, an adduct of dibenzofulvene with amine, which is a deprotecting reagent, (hereinafter to be sometimes referred to as “dibenzofulvene amine adduct” in the present specification) is by-produced. Particularly in the peptide synthesis, the dibenzofulvene amine adduct needs to be efficiently removed, since it possibly causes side reactions such as 9-fluorenylmethylation and the like, when the dibenzofulvene amine adduct remains in the next step. However, the dibenzofulvene amine adduct is highly liposoluble, and cannot be removed by washing with water and the like of the reaction mixture.
In peptide synthesis, the Fmoc group is mainly utilized as a protecting group for a solid phase synthesis method, which can easily remove a dibenzofulvene amine adduct by washing a solid support. However, the solid phase method has problems in scaling up and reactivity, since the reaction is limited to the surface of a solid support.
On the other hand, a Boc (tert-butoxycarbonyl) group capable of removing a by-product of deprotection as a gas (isobutene, carbon dioxide) is mainly utilized for a liquid phase peptide synthesis method, since dibenzofulvene amine adduct cannot be easily removed. However, when a peptide containing a sulfur-containing amino acid such as cysteine, methionine and the like is to be synthesized, what is called a Boc method, wherein a Boc group is used at the N-terminal and a Z group is used in combination for the protection of a functional group of an amino acid side chain or the C-terminal, cannot be employed, since the sulfur-containing amino acid poisons catalysts and the Z group cannot be deprotected with a catalytic reduction. Thus, use of the Fmoc group as an N-terminal protecting group is sometimes desired.
In view of such background, development of a liquid phase method permitting easy scaling up and suitable for industrial production of peptide pharmaceutical products and the like is desired, which can efficiently remove dibenzofulvene amine adduct when the Fmoc group is used as a protecting group.
Jikken Kagaku Koza fifth edition, (Japan), MARUZEN CO., LTD., published on Mar. 31, 2005, vol. 16, page 272, describes a method for removal of a dibenzofulvene amine adduct in the liquid phase peptide synthesis, which includes adding a hydrocarbon solvent such as hexane and the like for trituration of a residue obtained by concentrating a reaction extract to dryness, thereby dissolving a dibenzofulvene amine adduct in the solvent, and isolating the deprotected peptide as crystals.
However, this method is poor in operability, sometimes fails to reproduce at a large scale, and is unsuitable for industrial production. In addition, when a desired deprotected peptide is an oily substance, this method cannot be used, since it requires crystallization of the peptide. Furthermore, the method is associated with problems of low recovery rate and the like due to dissolution of peptide itself in a hydrocarbon solvent when the peptide chain is short.